Galgo: a bi-objective evolutionary meta-heuristic identifies robust transcriptomic classifiers associated with patient outcome across multiple cancer types.

MOTIVATION: Statistical and machine-learning analyses of tumor transcriptomic profiles offer a powerful resource to gain deeper understanding of tumor subtypes and disease prognosis. Currently, prognostic gene-expression signatures do not exist for all cancer types, and most developed to date have been optimized for individual tumor types. In Galgo, we implement a bi-objective optimization approach that prioritizes gene signature cohesiveness and patient survival in parallel, which provides greater power to identify tumor transcriptomic phenotypes strongly associated with patient survival. RESULTS: To compare the predictive power of the signatures obtained by Galgo with previously studied subtyping methods, we used a meta-analytic approach testing a total of 35 large population-based transcriptomic biobanks of four different cancer types. Galgo-generated colorectal and lung adenocarcinoma signatures were stronger predictors of patient survival compared to published molecular classification schemes. One Galgo-generated breast cancer signature outperformed PAM50, AIMS, SCMGENE and IntClust subtyping predictors. In high-grade serous ovarian cancer, Galgo signatures obtained similar predictive power to a consensus classification method. In all cases, Galgo subtypes reflected enrichment of gene sets related to the hallmarks of the disease, which highlights the biological relevance of the partitions found. AVAILABILITY AND IMPLEMENTATION: The open-source R package is available on www.github.com/harpomaxx/galgo. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Estrogen-dependent Leydig cell protein recognized by monoclonal antibody to MCF-7 cell line.

A protein (27,000 molecular weight) was previously found in rat Leydig cells after treatment with estradiol (E2) and human chorionic gonadotropin (hCG) in vitro. The effect of hCG occurred through increased E2 production. This hormone-regulated rat testicular protein was compared to an estrogen-regulated protein of similar physical characteristics isolated from a human mammary cancer cell line (MCF-7) and present in normal human estrogen target organs. The Leydig cells from rat and human tissue showed specific immunofluorescence and immunoperoxidase staining in the cytoplasm upon incubation with a monoclonal antibody (C11) to the estrogen-regulated protein from MCF-7 cells. Leydig cells after exposure to E2 or hCG showed the highest fluorescence intensity; this intensity was reduced by treatment with Tamoxifen. No reaction was associated with other testicular cells. The estrogen-regulated protein from human cell lines is therefore immunologically similar to that from the rat Leydig cell. The monoclonal antibody should be useful for further characterization of the Leydig cell protein.

Biological and clinical implications of heat shock protein 27,000 (Hsp27): a review.

Heat shock and other environmental and pathophysiologic stresses stimulate synthesis of heat shock proteins (Hsps). These proteins enable the cell to survive and recover from stressful conditions by as yet uncompletely understood mechanisms. Hsp27 is an important small Hsp (molecular weight, 27,000) found in human cells--both cancer cells and normal cells. This protein, besides its putative role in thermotolerance, is of special clinical interest because of recent data suggesting it may also play a role in drug resistance. In adults, Hsp27 is found particularly in several cell types such as breast, uterus, cervix, placenta, skin, and platelets. Although low-molecular-weight (small) Hsps have been found to be involved in embryogenesis of Xenopus and Drosophila, they have not been detected in human fetal organs. Regulation of expression of the Hsp gene (also known as HSPB1) has been considered a paradigm of gene regulation and is actively being studied in both prokaryotes and eukaryotes. In prokaryotes, the major Hsp genes are transcriptionally regulated by positively and negatively acting transcription factors. In eukaryotes, the genes encoding Hsps contain a regulatory DNA motif (inverted repeats of the pentameric sequence nGAAn) known as the heat shock element. Hsp27 may function as a molecular chaperone and in signal transduction pathways of different cell regulators, and Hsp27 and other Hsps may be active in development of resistance to stressful conditions and agents including cytotoxic drugs. Study findings indicate that some but not all estrogen-positive breast cancers express Hsp27, and overexpression of Hsp27 has been associated with both good and poor prognosis. In endometrial carcinomas, the presence of Hsp27 is correlated with the degree of tumor differentiation as well as with the presence of estrogen and progesterone receptors. Studies suggest, however, that detection of Hsp27 should not be considered to be a method for identifying hormone-responsive tumors or detecting estrogen receptors. Hsp27 seems to be a biochemical marker of estrogenic endometrial response. In patients with cervical cancer, Hsp27 is predominantly expressed in well-differentiated and moderately differentiated squamous cell carcinomas. In addition, expression of Hsp27 seems to be a negative prognostic factor for gastric cancer. Different isoforms of Hsp27 have been found in lymphoid tissue of patients with acute lymphoblastic leukemia, and the protein has also been associated with viral infections. These aspects are summarized and discussed in the present review.

Heat shock protein hsp70 in patients with axillary lymph node-negative breast cancer: prognostic implications.

BACKGROUND: Cell synthesis of heat shock (stress-response) proteins is increased by a variety of environmental and pathophysiological stressful conditions. The 70-kd heat shock protein (hsp70) is thought to be involved in protein-protein interactions including those of the protein products of the human c-myc oncogene and the p53 (also known as TP53) tumor suppressor gene. PURPOSE: The purpose of this study was to investigate whether elevated hsp70 expression may be an indicator of biological stress experienced by a breast cancer and may, therefore, predict disease outcome. METHODS: Levels of hsp70 were determined by Western blot analysis in primary breast tumors from patients with negative axillary lymph nodes. We performed exploratory data analyses on a set of 162 primary breast cancers and constructed prognostic indexes of hsp70 expression levels. The optimal cutpoint for hsp70 expression was considered to be the value yielding the greatest separation for disease-free survival for the resulting two groups of patients. That cutpoint was then validated in a set of 345 tumors by univariate and multivariate analyses. Data were analyzed for overall survival, disease-free survival, tumor size, and patient age, as well as estrogen receptor and progesterone receptor status, ploidy (DNA content), and percentage of cells in S phase as determined by flow cytometry. RESULTS: Expression of hsp70 emerged as a useful prognostic factor, both in univariate and in multivariate analyses. Patients whose tumors had high expression of hsp70 had significantly shorter disease-free survival (P = .006). The other statistically significant factors were S-phase fraction (P = .008) and tumor size (P = .01). For patients who received adjuvant therapy, hsp70 was the only independent predictor of disease recurrence (P = .05). For those with tumors 1-3 cm in diameter, hsp70 (P = .008) and S-phase fraction (P = .02) were statistically significant predictors of recurrence. CONCLUSIONS: Measurement of hsp70 expression in primary tumors from patients with node-negative breast cancer may be useful in identifying patients at high risk for disease recurrence and thus may affect decisions regarding treatment after surgery. IMPLICATIONS: Future studies should be performed to determine if detection of hsp70 by immunohistochemistry can be used to predict clinical outcome and to better understand the relationships between hsp70 and the effects of various treatment modalities.

Study of estrogen receptor, progesterone receptor, and the estrogen-regulated Mr 24,000 protein in patients with carcinomas of the endometrium and cervix.

The presence of an estrogen-regulated protein with 24,000 molecular weight has been studied in 47 patients with endometrial carcinomas and in 29 patients with cervical carcinomas in order to correlate its presence with that of estrogen receptors (ERs) and progesterone receptors (PgRs). In the cytosol tumor samples the Mr 24,000 protein was detected by the Western blot technique using a monoclonal antibody (C11), while the presence of ER and PgR was studied by the one-point dextran-coated charcoal assay. In the tumor tissue sections immunohistochemistry was applied to detect Mr 24,000 protein, ER, and PgR; in these cases monoclonal antireceptor antibodies (H222 and mPRI) were used to localize the receptor proteins. In endometrial and endocervical adenocarcinomas the presence of Mr 24,000 protein correlated significantly with that of ER (P less than or equal to 0.05) in the cytosol samples; when the evaluation was performed in the tumor sections, the presence of Mr 24,000 protein correlated with that of ER (P less than or equal to 0.005) and PgR (P less than or equal to 0.05) as well. The study also showed that almost 70% of the well-differentiated adenocarcinomas had ER, PgR, and Mr 24,000 protein. In 25% of the endometrial adenocarcinomas examined the tumors were associated with normal, proliferative, and hyperplastic endometrium; in these cases the presence of ER, PgR, and Mr 24,000 protein was evaluated by immunohistochemistry in the malignant and nonmalignant endometrium. On the other hand, there was a lack of correlation between Mr 24,000 protein, ER, and PgR in the squamous carcinomas of the uterine cervix and in the endometrial adenocarcinomas with squamous cells. In most of these cases the tumors lacked ER and PgR although 80% of them contained the Mr 24,000 protein to a variable degree. It is suggested that Mr 24,000 protein is involved in growth and differentiation (the Mr 24,000 protein is a heat shock protein) and that the gene coding of this protein is under hormonal control only in those tissues where growth and differentiation are strongly hormonally controlled (breast and endometrium).

Immunohistochemical detection of an estrogen-regulated protein by monoclonal antibodies.

It has been shown previously that estradiol regulates the synthesis of a Mr 24,000 cytosol protein in MCF-7 human breast cancer cells. This protein is an attractive probe to study the mechanisms of estrogen regulation in human breast cancer. We have produced monoclonal antibodies against the Mr 24,000 protein which show immunocytochemical localization in the cytoplasm of MCF-7 cells grown as solid tumors in nude mice. They do not react with normal mouse tissue or with MDA-MB-231 cells, an estrogen receptor-negative cell line. Moreover, the monoclonal antibodies react against certain human breast tumor biopsy samples. These data suggest that the monoclonal antibodies obtained will be useful to detect the estrogen-regulated protein by immunohistochemistry.

Response of human breast cancer cells to heat shock and chemotherapeutic drugs.

Previous studies have shown that certain chemotherapeutic drugs are less effective on tumor cells when cells have been previously exposed to hyperthermia. In the present study, we have evaluated whether specific modifications in heat shock protein (hsp) expression are associated with resistance to anticancer drugs. RNA levels for hsp90, hsp70, and hsp27 were studied by Northern and slot blots, while proteins were studied by two-dimensional gel electrophoresis, in MCF-7/BK and MDA-MB-231 breast cancer cells. The sensitivities of these cells to doxorubicin, colchicine, 5-fluorouracil, cisplatin, actinomycin D, and methotrexate were tested by clonogenic assays. These techniques were applied to both cell lines before (control) and after heat shock. The study revealed that elevated hsp70 and hsp27 levels were associated with doxorubicin resistance. In addition, the presence of phosphorylated hsp27 isoforms was also associated with doxorubicin resistance. The study showed that elevated hsps were not associated with multidrug resistance. Heat shock did not induce P170 glycoprotein mRNA overexpression or resistance to the other drugs tested. We also found that the level of doxorubicin protection conferred by the overexpression of hsp was lower than that obtained in cells expressing a multidrug resistance phenotype (MDA-A1R cells). In these cells, heat shock did not confer additional doxorubicin resistance and hsp27 phosphorylation was deficient. Our studies suggest that specific hsps are associated with doxorubicin resistance in certain human breast cancer cells and that this mechanism seems to be independent of the multidrug resistance system.

Tamoxifen and the isomers of 4-hydroxytamoxifen in tamoxifen-resistant tumors from breast cancer patients.

PURPOSE: The antiestrogen tamoxifen is effective in therapy for breast cancer. However, its use is limited by the eventual development of acquired tamoxifen resistance in many patients. The mechanisms responsible for tamoxifen resistance remain unknown; loss of estrogen receptor (ER), selection of hormone-independent breast cancer clones, or alterations in serum tamoxifen levels after long-term use do not explain acquired resistance in most patients. Using an experimental model in which human breast cancer cells develop resistance in athymic mice treated with tamoxifen, we have recently shown that acquired resistance is associated with markedly reduced cellular concentrations of tamoxifen and by isomerization of the trans-4-hydroxy metabolite to the less potent cis isomer. MATERIALS AND METHODS: Using a sensitive high-performance liquid chromatography (HPLC) assay, we have now measured levels of tamoxifen and its major metabolites in a series of 14 tumors from patients treated with tamoxifen. The duration of therapy ranged from 1 month to 6 years. RESULTS: Tumor tamoxifen levels varied over a wide range. Low concentrations were observed in tumors from eight patients, all demonstrating progressive disease at the time of biopsy after a minimum duration of treatment of 6 months. Six tumors had moderate to high tamoxifen levels, two from patients responding to tamoxifen, one from a patient with stable disease, and three from patients with disease progression. Both the cis and trans isomers of the potent antiestrogenic metabolite 4-hydroxy-tamoxifen were detected in 11 tumors. Six tumors had high ratios of the cis to trans isomer (1.10:2.06), all from patients not responding to tamoxifen. The five tumors with low cis:trans ratios included the two tumors from responding patients and three from patients with progression. All but one of the 11 nonresponding patients had either a low tumor tamoxifen level, a high cis:trans ratio, or both. CONCLUSION: This study clearly demonstrates a wide range of tumor tamoxifen levels and accumulation of the less antiestrogenic cis isomer of 4-hydroxytamoxifen in some patients on tamoxifen therapy. Additional study is necessary to determine if these metabolic profiles are related to the development of tamoxifen resistance.

Estrogen receptors and cell proliferation in breast cancer.

Most of the actions of estrogens on the normal and abnormal mammary cells are mediated via estrogen receptors (ERs), including control of cell proliferation; however, there are also alternative pathways of estrogen action not involving ERs. Estrogens control several genes and proteins that induce the cells to enter the cell cycle (protooncogenes, growth factors); estrogens also act on proteins directly involved in the control of the cell cycle (cyclins), and moreover, estrogens stimulate the response of negative cell cycle regulators (p53, BRCA1). The next challenge for researchers is elucidating the integration of the interrelationships of the complex pathways involved in the control of cell proliferation. This brief review focuses on the mechanisms of estrogen action to control cell proliferation and the clinical implications in breast cancer. (Trends Endocrinol Metab 1997;8:313-321). (c) 1997, Elsevier Science Inc.

The small heat shock protein HSP27 is not an independent prognostic marker in axillary lymph node-negative breast cancer patients.

Heat shock protein 27 (hsp27) belongs to the family of heat shock proteins and is thought to be involved in thermotolerance, cell proliferation, drug resistance, and chaperone processes. The aim of this study was to investigate whether hsp27 levels are correlated with clinical outcome in axillary lymph node-negative breast cancer patients. We describe a Western blot study measuring hsp27 levels in 425 patients and an immunohistochemistry (IHC) study analyzing 788 patients. Results obtained by both methods were concordant. Univariate survival analysis was performed considering hsp27 either as an optimally dichotomized variable or as a continuous variable. Additional data include age at biopsy, tumor size, estrogen receptor (ER) and progesterone receptor status, tumor ploidy and percentage of cells in S phase, and adjuvant therapy. hsp27 levels correlated positively with ER status (P = 0.0001 in Western blot and IHC study), progesterone receptor status (P = 0.0001 in Western blot and IHC study), and aneuploidy (Western blot study, P = 0.0012; IHC study, P = 0.0004) but not with tumor size (Western blot study, P = 0.69; IHC, P = 0.53) or S phase (Western blot study, P = 0.19; IHC study, P = 0.38). Overall, there was no relationship between hsp27 expression and disease-free survival (Western blot study, P = 0.70/0.54; IHC, P = 0.47/0.30) or overall survival (Western blot study, P = 0.16/0.15; IHC, P = 0.46/0.78). Exploratory subset analyses defined by ER status and use of adjuvant treatment indicated that in ER+/untreated patients, high hsp27 levels correlated modestly with shorter disease-free survival (Western blot, P = 0.04/0.04; IHC, P = 0.11/0. 03). hsp27 is not a useful prognostic marker for the clinic in axillary lymph node-negative patients. However, the finding of modest prognostic value of hsp27 in the subgroup of ER+/untreated patients raises new questions about the biological function of hsp27 in breast cancer.

Heat shock proteins hsp27 and hsp70: lack of correlation with response to tamoxifen and clinical course of disease in estrogen receptor-positive metastatic breast cancer (a Southwest Oncology Group Study).

In this study, we tested the hypothesis that heat shock proteins (hsps) 27 and 70 are associated with clinical resistance to tamoxifen. hsp27 is, like progesterone receptor, an estrogen-regulated protein. hsp70 is also of interest because of its interaction with estrogen receptors and because hsp70 is a component of the molecular chaperone machinery functioning in the assembly and trafficking of steroid receptors. In addition, hsps in general help protect cells against noxious stimuli and stress, and their expression has been linked to drug resistance. The study involved 205 tumors from estrogen receptor-positive tamoxifen-treated breast cancer patients with metastatic disease. All patients received daily tamoxifen as initial therapy for metastatic disease. The study began in 1982, and follow-up is now 9 years. hsp27 and hsp70 were detected by immunohistochemistry and scored according to the nuclear and/or cytoplasmic content. Expression of hsp27 or hsp70 was unrelated to estrogen receptor content, progesterone receptor content, menopausal status, age, and presence of visceral disease. Cytoplasmic and nuclear hsp27 positivities were weakly and inversely related to each other (P = 0.05). There was a significant association between cytoplasmic hsp27 and cytoplasmic hsp70 content (P < 0.001), as well as between nuclear hsp70 and nuclear hsp27 content (P = 0.001). Cytoplasmic and nuclear hsp70 were also associated (P = 0.02). However, increased hsp27 and hsp70 expression (nuclear or cytoplasmic) was not significantly associated with response to tamoxifen, time to treatment failure, or survival. Thus, this study clarifies the lack of clinical utility of hsp27 and hsp70 in predicting the response to tamoxifen in an estrogen receptor-positive breast cancer population.

Serotonin secretion from rat Leydig cells.

In rat Leydig cells, serotonin (5HT) binds to 5HT2 receptors and stimulates the secretion of CRF which in turn acts as an inhibitor of gonadotropin-induced cAMP formation and androgen production. In the present study we defined the regulation of 5HT secretion in cultured Leydig cells. Adult Leydig cells secreted considerable quantities of 5HT (100-150 pg/10(6) cells per 10 min). The release of 5HT was acutely stimulated by hCG (ED50, 1.1 pM) with maximal stimulation at 10 pM hCG (160%). Forskolin also increased (+220%) 5HT release from cultures (ED50, 50 nM) while TPA was much less effective (+20%), indicating a major role for cAMP in gonadotropin-induced 5HT release. This was confirmed by the finding that 8-Br cAMP (1 mM) was an effective stimulus of 5HT release (+360%). Similar increases of 5HT release by hCG were observed in the absence of extracellular Ca2+. However, ionomycin was a potent stimulus of 5HT release, indicating that elevation of cytoplasmic [Ca2+] could also induce amine secretion. The 5HT content of Leydig cells ranged from 300 to 350 pg/10(6) cells, and decreased during stimulation of 5HT release. Also, immunohistochemical studies revealed specific staining of 5 HT in interstitial cells of the adult rat testis. These studies demonstrated that rat Leydig cells contain and secrete 5HT, and that 5HT release is stimulated by gonadotropin acting primarily through a cAMP-mediated mechanism.

Growth hormone-releasing hormone in testicular interstitial and germ cells: potential paracrine modulation of follicle-stimulating hormone action on Sertoli cell function.

GH-releasing hormone (GHRH) is present in the interstitial and germ cells of the rat testis. In previous studies we found that GHRH is secreted from rat adult Leydig cells, in which it stimulates basal and LH-induced cAMP formation and steroidogenesis. In other studies cAMP production in Sertoli cells was found to be stimulated by GHRH. In the present report, we describe a potential paracrine action of GHRH in the Sertoli cell, with stimulation of cAMP formation in cultured adult and pubertal Sertoli cells. GHRH increased FSH-stimulated cAMP production in adult and pubertal cultures in a time-dependent manner. GHRH stimulation of basal and FSH-induced extracellular cAMP formation was more prominent in pubertal than in adult cultures. Immunocytochemical studies demonstrated the presence of GHRH-like immunoreactivity in rat interstitial cells from day 4 to adult life and in the acrosomal region of early and intermediate spermatids at stages III-VI of the seminiferous epithelium cycle. Immunoreactive GHRH was not observed in late spermatids and mature sperm or in Sertoli cells at any age. These results indicate that GHRH acts synergistically with FSH to promote cAMP production in Sertoli cells in culture. Testicular GHRH of Leydig and germ cell origin may be an important paracrine regulator of Sertoli cell function. Alternatively, GHRH present in germ cells may exert stage-specific intracrine functions.

Identification of seven hormone-producing cell types in the human pharyngeal hypophysis.

Eight adult human pharyngeal pituitary glands taken at autopsy were studied by immunocytochemistry to reveal the presence of ACTH-, lipotropin-, FSH-, LH-, TSH-, PRL-, and GH-immunoreactive cells. All of these cell types were found and quantitated in pharyngeal hypophyses from patients with no evidence of endocrine disorder. The percentage of the seven hormone-producing cell types varied from gland to gland from 1-30%; there were no marked histological differences between sexes or attributable to age. The cellular composition of the pharyngeal hypophysis shows that this gland has the capacity to produce at least seven hormones.

The presence of an estrogen-regulated protein detected by monoclonal antibody in abnormal human endometrium.

The cellular localization of a 24,000 (24K) mol wt protein was evaluated by monoclonal antibody immunocytochemistry in atrophic and persistent proliferative endometrium as well as in various forms of hyperplastic and neoplastic human endometria. 24K was chiefly located in the cytoplasm of ciliated cells and their precursor forms, clear cells. It was absent in atrophic, resting, and regressive forms of cystic glandular hyperplasia. Persistent proliferative endometria with a low degree of proliferation has occasional 24K immunostained cells, whereas those with active proliferation contained intense 24K immunostaining. A similar pattern characterized the active forms of cystic glandular hyperplasia. The immunostaining reaction decreased in adenomatous and atypical adenomatous hyperplasia as well as in well differentiated adenocarcinomas. In poorly differentiated carcinomas, 24K protein was virtually absent. These findings suggest that 24K protein is estrogen and ciliated cell related, and endometrial ciliogenesis and 24K protein are morphological and biochemical markers, respectively, of the estrogenic endometrial response. Their increase and decrease in hyperplastic and neoplastic endometria support the concept that early hyperplasia is highly sensitive to estrogenic stimulation, whereas with increasing architectural and cytologic atypia, the estrogenic response decreases as a reflection of growing cell populations that are independent of estrogenic influence. Immunostaining with 24K is not sensitive enough to discriminate estrogen-independent cells of atypical adenomatous hyperplasia from those of early, well differentiated adenocarcinoma.

Evidence for modulation of a 24K protein in human endometrium during the menstrual cycle.

The presence and distribution of a protein with a molecular weight of 24,000 (24K) was determined in endometrial biopsies from regularly cycling women. This protein, of as yet unknown function and originally found in a breast cancer cell line, was detected by immunohistochemistry using a monoclonal antibody. In the glandular epithelium, the 24K protein began to appear during the late proliferative phase and decreased after ovulation. In contrast, the strongest immunostaining was observed in the superficial epithelium during the secretory phase. In these cells, the 24K immunoactivity reached the maximum around day 21 of the cycle and was clearly seen in the bulbous projections of the apical cytoplasm. These results suggest that the 24K protein may be a marker for hormonal events during the menstrual cycle.

Serological detection of heat shock protein hsp27 in normal and breast cancer patients.

Heat shock protein Mr 27,000 (hsp27) is found in many human breast cancer cells and tissues; its expression is associated with the presence of estrogen receptors, lower cell proliferation, and resistance to certain chemotherapies. The purpose of this study was to assess whether hsp27 may be present in sera from women with primary breast cancer and to know whether autoantibodies to hsp27 may be found in these patients. The study was performed by Western blot analyzing sera from 42 normal premenopausal women, 20 normal postmenopausal women, and 36 breast cancer patients. hsp27 was clearly detected in sera by immunoblotting but only after immunoprecipitation. The mean hsp27 levels in cancer patients were higher than in the control patients; however, 66% of the breast cancer patients showed hsp27 within the normal range, indicating low sensitivity. Moreover, cancer patients with metastatic disease did not show significantly higher hsp27 levels than cancer patients without metastases. Serum hsp27 levels did not correlate with the hsp27 levels in tumor tissues detected by immunohistochemistry. Elevated CA 15-3 levels were not associated with high hsp27 values. Autoantibodies against hsp27 were not detected by immunoblotting in normal sera and in sera from breast cancer patients. As a consequence, serological determination of this biomarker is unlikely to be of utility in the detection and follow-up of breast cancer patients.

Improved detection of apoptotic cells using a modified in situ TUNEL technique.

We are in the process of assessing the response of cancer tissues to chemotherapy, evaluating, among other points, the proportion of cancer cells undergoing apoptosis. However, the apoptotic index obtained with the original TUNEL technique was lower than that obtained by evaluation of apoptosis on H&E-stained sections. Here we describe a small modification of the TUNEL technique that significantly increases the sensitivity of the assay. In the nonmodified TUNEL technique, a digoxigenin-labeled probe is detected using a direct peroxidase-conjugated system, whereas here we report the advantage of using a streptavidin-biotin-immunoperoxidase system. This, in conjunction with pretreatment of tissue sections with proteinase K and microwave irradiation, improved the detection of apoptotic cells.

Intensification of the immunocytochemical reaction by staining both sides of tissue sections.

We describe how the efficiency of immunostaining may be increased by staining paraffin sections on both sides. This modification exposes more antigenic binding sites per unit tissue area, as shown by the peroxidase-antiperoxidase and the avidin-biotin-peroxidase complex methods. Using antigen-rich tissue samples, the modified procedure made it possible to use more dilute primary antiserum or to reduce the incubation time of the tissue with the primary antibody. Alternatively, in tissue samples with sparse antigenic sites, the procedure made it possible to visualize and document very weak immunoreactivities.

A silver staining method for single-cell gel assay.

The single-cell gel assay (comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. Usually, the comets are visualized and evaluated with fluorescent DNA stains. This staining requires specific equipment (e.g., a high-quality fluorescence microscope), the slides must be analyzed immediately, and they cannot be stored for long periods of time. Here we describe, using human lymphocytes, some modifications of the silver staining for comets that significantly increase the sensitivity/reproducibility of the assay. This silver staining was compared with fluorescence staining and commercial silver stains. (J Histochem Cytochem 49:1183-1186, 2001)